Membrane proteins are the major group of targets for antibody therapeutics. Integral membrane proteins, such as G protein-coupled receptors (GPCRs), ion channels and transporters, are involved in diverse biological functions and also in many diseases. Approximately 40% of all modern medical drugs target GPCRs. However, due to their hydrophobic domains, membrane proteins are difficult to solubilize and to purify. Pure and stable protein samples of membrane proteins are hardly available. Therefore there is high need to provide technologies for the efficient presentation of membrane proteins in order to develop novel antibody-based therapeutics.
Retroviruses are enveloped particles of around 100 nm in size (Human Immunodeficiency Virus (HIV): ˜120 nm, Moloney Murine Leukemia Virus (MoIMLV): ˜90 nm). The virions contain two identical single-stranded RNA molecules of 7-10 kb in length. The envelope of the virus is acquired during the assembly of the virus at the plasma membrane and contains host cell phospholipids and proteins, but also some viral glycoproteins (in the case of HIV for example gp41 and gp120). The role of these viral envelope proteins is to identify and bind to receptor sites on target cells. After binding, the virus fuses with the target cell membrane, allowing the capsid and the viral genome to enter and infect the target/host cell. After infection of the host cell the virus uses its own reverse transcriptase to synthesize DNA from its RNA genome. The integrase enzyme inserts the DNA into the host cells genome. Afterwards, the host cell expresses the viral proteins required for assembly of new copies of the virus.
As one example, the HIV DNA contains 3 main genes (gag, pol, env) and 6 accessory genes (vif, vpr, vpu, tat, rev, nef). The three main genes contain information needed to make the structural proteins for new virus particles. ENV encodes the viral envelope protein gp160, which is cleaved by furin to form gp120 and gp41. These are transported to the plasma membrane of the host cell, where gp41 anchors gp120 to the membrane of the infected cell. POL encodes the enzymes required for replication (reverse transcriptase) and integration (integrase) of the virus as well as a viral protease. Gag is a multi-domain polyprotein. The Gag polyprotein is cleaved by the viral protease separating six proteins: the three folded domains matrix (MA), capsid (CA) and nucleocapsid (NC) and three shorter peptides SP1, SP2 and p6. The accessory genes encode for regulatory proteins that control the ability of HIV to infect cells, produce new copies of virus (replicate), or cause disease.
The assembly of the virus occurs at the host cell plasma membrane, into which the viral envelope proteins were inserted. The gag proteins, the major part of the viral capsid (2000-4000 copies per virion), associate with the inner surface of the plasma membrane. Gag recruits other essential virion components including the viral replication proteins (expressed as Gag-Pol fusion proteins) and the genomic RNA. Assembly of the Gag proteins leads to the budding of the virus, which is initially a noninfectious virion. This so-called immature virion mainly contains uncleaved Gag polyproteins. Formation of an infectious virion requires processing of Gag by the viral protease at five specific sites, leading to a rearrangement of the interior organization.
Virus-like particles (VLPs) resemble viruses but are non-infectious because they do not contain any viral genetic material. The expression of viral structural proteins results in the self-assembly of virus like particles (VLPs). Different strategies exist for the generation of VLPs. VLPs can be produced in a variety of cell culture systems including mammalian cell lines, insect cell lines, yeast, and plant cells.
Principally, the recombinant over-expression of Gag is sufficient for the formation of virus-like particles. The Gag proteins associate with the inner surface of the plasma membrane causing the budding of empty shells into the cell culture medium. The empty shell is surrounded by plasma membrane of the host cell and contains various host cell proteins.
Viral major capsid proteins, such as the HIV GAG-protein, are known tools for the generation of virus-like particles (VLPs) (Delachambre, 1989, Deml 1997). Such VLPs can be used for the expression of heterologous proteins, in particular proteins which are difficult to express by other means, for example membrane spanning protein. See for example U.S. Pat. No. 7,763,258. However, these systems are characterized by the co-expression of the viral capsid protein and the heterologous membrane spanning protein.
Viral major capsid proteins have also been used to generate vaccines. See for example EP0449116 or WO07/054792. In such approaches, the capsid proteins were modified to incorporate certain heterologous proteins which are presented to the immune system on said capsid proteins. Also bacteriophage coat proteins were used to generate antigen-presenting phage-derived particles for vaccination (WO06/032674). However, such phage-derived particles are produced in E. coli, and are therefore not surrounded by plasma membrane of a eukaryotic cell.
Virus-like particles have also been generated utilizing other proteins than viral capsid proteins for the presentation of peptides or proteins on the virus-like particles. Such proteins include neuraminidase (NA) and hemmagglutinin (HA) (Kaczmarczyk et al. (PNAS) 108, 16998-17003).
Other systems utilize protein-protein interaction techniques in which both, the capsid protein and the protein of interest, are fused to a partner peptidic or proteinaceous moiety, which interact with each other. This leads to the co-localization of the capsid protein with the protein of interest, and hence the protein of interest may be presented or displayed on a VLP. See Mayr & Schenker (presentation on the 8th PEGS Annual Conference, Boston, Apr. 30-May 3, 2012).
The present disclosure provides an improved method for the presentation and display of proteins, in particular, transmembrane proteins, on virus-like particles.
Importantly, the present disclosure makes use of fusion proteins, in which the protein that is to be displayed is N-terminally fused to the viral capsid protein. Viral capsid proteins are N-terminally myristoylated, and therefore the prior art did not use such N-terminal fusions. In the present disclosure it is shown, that such fusion proteins can however be successfully expressed and the respective protein of interest be presented in virus-like particles. Such a system has numerous advantages.